Novel ELISA for the diagnosis of histoplasmosis

TOSCANINI MA; BRITO DEVOTO T; IOVANNITTI C; NUSBLAT AD; CUESTAS ML

Congreso; 17TH INFOCUS; 2019

Objectives: Histoplasmosis is a systemic and endemic mycosis caused by Histoplasma capsulatum. The significance of histoplasmosis results from its worldwide distribution, its ability to mimic other serious disease entities and its propensity to cause serious disseminated infection in immunocompetent patients. Definitive diagnosis involves direct visualization of the fungus in tissue and/or by culture, which may take up to 4 weeks and lacks sensitivity. The detection of antibodies offers a rapid alternative to microbiological means of diagnosis, and their detection by immunodiffusion (ID) and complement fixation (CF) is often used. Histoplasmin, the standard serodiagnostic reagent used is not ideal because of the presence of cross-reactive carbohydrate components which lead to a detrimental effect on the specificity and sensitivity of the CF and ID tests, respectively, particularly in the early acute stage of the disease. Moreover, current methods for its production are time-consuming and problematic. An alternative approach to immunodiagnosis is to detect fungal antigens in urine or serum, which is particularly useful in cases of disseminated disease. In this study, the application of a novel antigen, the 100kDa protein of H. capsulatum, Hcp100, for the development of an ELISA for the early diagnosis of histoplasmosis is described. Methods: Serum samples from 15 histoplasmosis patients were studied. Five of them had the chronic form, one the disseminated form and nine had AIDS and the disseminated form of histoplasmosis. The diagnosis for all individuals was confirmed either by culture or histopathology. Only the AIDS patients studied were serology (ID) negative. Serum samples from patients with paracoccidioidomycosis, coccidioidomycosis, cryptococcosis, tuberculosis and pneumocystosis were included. As negative controls, 100 serum samples from healthy individuals were used. For the development of the novel ELISA, the gene that encodes for Hcp100 was expressed in the Pichia pastoris X-33 strain as a soluble protein, purified by affinity chromatography and used at a concentration of 0.5 µg/well for coating the ELISA plate. After blocking with 5% (w/v) dried non-fat milk in PBS-Tween20 0.05%, serum samples were added at a dilution of 1:1000 and incubated for 1h at room temperature. Then, goat anti-human IgG horseradish peroxidase conjugate at a dilution of 1:10000 was used as a secondary antibody. The reaction was developed with OPD and terminated by addition of H2SO4 0.4N followed by absorbance measurement at 492nm. Sensitivity, specificity, predictive values and the area under the curve (ROC) were estimated. Results: ROC analysis determined the optimal cutoff for Hcp100 antibody detection at which point the sensitivity was 80% and the specificity was 86%. Noteworthy, 6 out of 9 AIDS patients tested positive by ELISA whilst none were positive by ID. Positive and negative predictive values were 39% and 97%, respectively.Conclusion: Detection of antibodies to Hcp100 has the potential to aid in the diagnosis of histoplasmosis, identifying cases that are falsely negative by ID. The ELISA developed herein can easily be adapted for use in-house in regional centers. Selected peptide epitopes will be useful in the development of more sensitive and specific diagnostic tests.